PURIFICATION AND CHARACTERIZATION OF MALEYLACETATE REDUCTASE FROM ALCALIGENES-EUTROPHUS JMP134(PJP4)

Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degrad ation of chloroaromatic compounds by channeling maleylacetate and some of its substituted derivatives into the 3-oxoadipate pathway. The enz yme was purified to apparent homogeneity from an extract of 2,4-dichlo rophenoxyacetate (2,4-D)-grown cells of Alcaligenes eutrophus JMP134. Maleylacetate reductase appears to be a dimer of two identical subunit s of 35 kDa. The pI was determined to be at pH 5.4. There was no indic ation of a flavin prosthetic group. The enzyme was inactivated by p-ch loromercuribenzoate but not by EDTA, 1,10-phenanthroline, or dithiothr eitol. Maleylacetate and 2-chloromaleylacetate were converted with sim ilar efficiencies (with NADH as cosubstrate, K(m) = 31 muM for each su bstrate and k(cat) = 8,785 and 7,280/min, respectively). NADH was pref erred to NADPH as the cosubstrate. Upon reduction of 2-chloromaleylace tate by the purified enzyme, chloride was liberated and the resulting maleylacetate was further reduced by a second NADH. These results and the kinetic parameters suggest that maleylacetate reductase is suffici ent to channel the 2,4-D degradation intermediate 2-chloromaleylacetat e into the 3-oxoadipate pathway. In a data base search the NH2-termina l sequence of maleylacetate reductase was found to be most similar to that of TfdF, a pJP4-encoded protein of as-yet-unknown function in 2,4 -D degradation.